OBJECTIVE

To determine whether low vitamin D concentrations are associated with an increased risk of incident all-cause dementia and Alzheimer disease.

METHODS

One thousand six hundred fifty-eight elderly ambulatory adults free from dementia, cardiovascular disease, and stroke who participated in the US population-based Cardiovascular Health Study between 1992-1993 and 1999 were included. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were determined by liquid chromatography-tandem mass spectrometry from blood samples collected in 1992-1993. Incident all-cause dementia and Alzheimer disease status were assessed during follow-up using National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association criteria.

RESULTS

During a mean follow-up of 5.6 years, 171 participants developed all-cause dementia, including 102 cases of Alzheimer disease. Using Cox proportional hazards models, the multivariate adjusted hazard ratios (95% confidence interval [CI]) for incident all-cause dementia in participants who were severely 25(OH)D deficient (<25 nmol/L) and deficient (≥25 to <50 nmol/L) were 2.25 (95% CI: 1.23-4.13) and 1.53 (95% CI: 1.06-2.21) compared to participants with sufficient concentrations (≥50 nmol/L). The multivariate adjusted hazard ratios for incident Alzheimer disease in participants who were severely 25(OH)D deficient and deficient compared to participants with sufficient concentrations were 2.22 (95% CI: 1.02-4.83) and 1.69 (95% CI: 1.06-2.69). In multivariate adjusted penalized smoothing spline plots, the risk of all-cause dementia and Alzheimer disease markedly increased below a threshold of 50 nmol/L.

CONCLUSIONS

Our results confirm that vitamin D deficiency is associated with a substantially increased risk of all-cause dementia and Alzheimer disease. This adds to the ongoing debate about the role of vitamin D in nonskeletal conditions.

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.

Intracellular pH (pHi) is an important modulator of cardiac excitation and contraction, and a potent trigger of electrical arrhythmia. This review outlines the intracellular and membrane mechanisms that control pHi in the cardiac myocyte. We consider the kinetic regulation of sarcolemmal H+, OH- and HCO3- transporters by pH, and by receptor-coupled intracellular signalling systems. We also consider how activity of these pHi effector proteins is coordinated spatially in the myocardium by intracellular mobile buffer shuttles, gap junctional channels and carbonic anhydrase enzymes. Finally, we review the impact of pHi regulatory proteins on intracellular Ca2+ signalling, and their participation in clinical disorders such as myocardial ischaemia, maladaptive hypertrophy and heart failure. Such multiple effects emphasise the fundamental role that pHi regulation plays in the heart.

The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D. In this study, topically applied 1,25(OH)(2)D(3), enhanced the suppressive capacity of CD4(+)CD25(+) cells from the draining lymph nodes. The effects of topical 1,25(OH)(2)D(3) were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)(2)D(3) production in skin. CD4(+) cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)(2)D(3)-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4(+)CD25(+) cells. Furthermore, purified CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated or UVB-irradiated mice compared with equal numbers of CD4(+)CD25(+) cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4(+)Foxp3(+) cells of donor origin significantly increased in recipients of CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)(2)D(3) may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.

BACKGROUND

Low plasma 25-hydroxyvitamin D (25[OH]D) concentration is associated with high arterial blood pressure and hypertension risk, but whether this association is causal is unknown. We used a mendelian randomisation approach to test whether 25(OH)D concentration is causally associated with blood pressure and hypertension risk.

METHODS

In this mendelian randomisation study, we generated an allele score (25[OH]D synthesis score) based on variants of genes that affect 25(OH)D synthesis or substrate availability (CYP2R1 and DHCR7), which we used as a proxy for 25(OH)D concentration. We meta-analysed data for up to 108 173 individuals from 35 studies in the D-CarDia collaboration to investigate associations between the allele score and blood pressure measurements. We complemented these analyses with previously published summary statistics from the International Consortium on Blood Pressure (ICBP), the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and the Global Blood Pressure Genetics (Global BPGen) consortium.

RESULTS

In phenotypic analyses (up to n=49 363), increased 25(OH)D concentration was associated with decreased systolic blood pressure (β per 10% increase, -0·12 mm Hg, 95% CI -0·20 to -0·04; p=0·003) and reduced odds of hypertension (odds ratio [OR] 0·98, 95% CI 0·97-0·99; p=0·0003), but not with decreased diastolic blood pressure (β per 10% increase, -0·02 mm Hg, -0·08 to 0·03; p=0·37). In meta-analyses in which we combined data from D-CarDia and the ICBP (n=146 581, after exclusion of overlapping studies), each 25(OH)D-increasing allele of the synthesis score was associated with a change of -0·10 mm Hg in systolic blood pressure (-0·21 to -0·0001; p=0·0498) and a change of -0·08 mm Hg in diastolic blood pressure (-0·15 to -0·02; p=0·01). When D-CarDia and consortia data for hypertension were meta-analysed together (n=142 255), the synthesis score was associated with a reduced odds of hypertension (OR per allele, 0·98, 0·96-0·99; p=0·001). In instrumental variable analysis, each 10% increase in genetically instrumented 25(OH)D concentration was associated with a change of -0·29 mm Hg in diastolic blood pressure (-0·52 to -0·07; p=0·01), a change of -0·37 mm Hg in systolic blood pressure (-0·73 to 0·003; p=0·052), and an 8·1% decreased odds of hypertension (OR 0·92, 0·87-0·97; p=0·002).

CONCLUSIONS

Increased plasma concentrations of 25(OH)D might reduce the risk of hypertension. This finding warrants further investigation in an independent, similarly powered study.

BACKGROUND

British Heart Foundation, UK Medical Research Council, and Academy of Finland.

BACKGROUND

In humans, few studies have compared the potencies of ergocalciferol and cholecalciferol in improving and maintaining vitamin D status.

OBJECTIVE

Our objective was to evaluate the effects of a single very large dose of both calciferols on serum changes of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D], ionized calcium, and parathyroid hormone (PTH) at baseline, and at 3, 7, 30, and 60 d.

METHODS

This was a prospective randomized intervention study.

METHODS

The study was performed in a nursing home residence.

METHODS

A total of 32 elderly female patients (age range 66-97 yr), with vitamin D deficiency was included in the study.

METHODS

Participants were randomized into four groups of eight to receive a single dose of 300,000 IU ergocalciferol or cholecalciferol by oral (os) or im route.

RESULTS

25(OH)D levels sharply increased at d 3 only when vitamins were given os. The 30-d basal difference in serum 25(OH)D was significantly greater after cholecalciferol os administration (47.8 +/- 7.3 ng/ml) compared with other forms (D(3) im: 15.9 +/- 11.3; D(2) os: 17.3 +/- 4.7; D(2) im: 5 +/- 4.4; all P < 0.001). The area under the curve (AUC) of the serum 25(OH)D against time (AUC(60)) was: D(3) os, 3193 +/- 759 ng x d/ml vs. D(2) os, 1820 +/- 512, P < 0.001; and D(3) im, 1361 +/- 492 vs. D(2) im, 728 +/- 195, P < 0.01. 25(OH)D significantly influences PTH levels at 3 (P < 0.03), 7 (P < 0.01), 30 (P < 0.01), and 60 d (P < 0.05). At 60 d, the form of vitamin (cholecalciferol) significantly lowers PTH levels (P = 0.037).

CONCLUSIONS

Cholecalciferol is almost twice as potent as ergocalciferol in increasing serum 25(OH)D, when administered either by mouth or im. 25(OH)D plays a role in modulating serum PTH.

BACKGROUND

Serum 25-hydroxyvitamin D (25-[OH]D) is considered the best biomarker of clinical vitamin D status.

OBJECTIVE

To determine the effect of increasing oral doses of vitamin D(3) on serum 25-(OH)D and serum parathyroid hormone (PTH) levels in postmenopausal white women with vitamin D insufficiency (defined as a 25-[OH]D level ≤50 nmol/L) in the presence of adequate calcium intake. These results can be used as a guide to estimate the Recommended Dietary Allowance (RDA) (defined as meeting the needs of 97.5% of the population) for vitamin D(3).

METHODS

Randomized, placebo-controlled trial. (ClinicalTrials.gov registration number: NCT00472823)

METHODS

Creighton University Medical Center, Omaha, Nebraska.

METHODS

163 healthy postmenopausal white women with vitamin D insufficiency enrolled in the winter or spring of 2007 to 2008 and followed for 1 year.

METHODS

Participants were randomly assigned to receive placebo or vitamin D(3), 400, 800, 1600, 2400, 3200, 4000, or 4800 IU once daily. Daily calcium supplements were provided to increase the total daily calcium intake to 1200 to 1400 mg.

METHODS

The primary outcomes were 25-(OH)D and PTH levels at 6 and 12 months.

RESULTS

The mean baseline 25-(OH)D level was 39 nmol/L. The dose response was curvilinear and tended to plateau at approximately 112 nmol/L in patients receiving more than 3200 IU/d of vitamin D(3). The RDA of vitamin D(3) to achieve a 25-(OH)D level greater than 50 nmol/L was 800 IU/d. A mixed-effects model predicted that 600 IU of vitamin D(3) daily could also meet this goal. Compared with participants with a normal body mass index (<25 kg/m(2)), obese women (≥30 kg/m(2)) had a 25-(OH)D level that was 17.8 nmol/L lower. Parathyroid hormone levels at 12 months decreased with an increasing dose of vitamin D(3) (P = 0.012). Depending on the criteria used, hypercalcemia occurred in 2.8% to 9.0% and hypercalciuria in 12.0% to 33.0% of participants; events were unrelated to dose.

CONCLUSIONS

Findings may not be generalizable to other age groups or persons with substantial comorbid conditions.

CONCLUSIONS

A vitamin D(3) dosage of 800 IU/d increased serum 25-(OH)D levels to greater than 50 nmol/L in 97.5% of women; however, a model predicted the same response with a vitamin D(3) dosage of 600 IU/d. These results can be used as a guide for the RDA of vitamin D(3), but prospective trials are needed to confirm the clinical significance of these results.

BACKGROUND

National Institute on Aging.

BACKGROUND

Vitamin D insufficiency is a common public health problem nationwide. Circulating 25-hydroxyvitamin D3 (25[OH]D), the most commonly used index of vitamin D status, is converted to the active hormone 1,25 dihydroxyvitamin D3 (1,25[OH]2D), which, operating through the vitamin D receptor (VDR), inhibits in vitro cell proliferation, induces differentiation and apoptosis, and may protect against prostate cancer. Despite intriguing results from laboratory studies, previous epidemiological studies showed inconsistent associations of circulating levels of 25(OH)D, 1,25(OH)2D, and several VDR polymorphisms with prostate cancer risk. Few studies have explored the joint association of circulating vitamin D levels with VDR polymorphisms.

RESULTS

During 18 y of follow-up of 14,916 men initially free of diagnosed cancer, we identified 1,066 men with incident prostate cancer (including 496 with aggressive disease, defined as stage C or D, Gleason 7-10, metastatic, and fatal prostate cancer) and 1,618 cancer-free, age- and smoking-matched control participants in the Physicians' Health Study. We examined the associations of prediagnostic plasma levels of 25(OH)D and 1,25(OH)2D, individually and jointly, with total and aggressive disease, and explored whether relations between vitamin D metabolites and prostate cancer were modified by the functional VDR FokI polymorphism, using conditional logistic regression. Among these US physicians, the median plasma 25(OH)D levels were 25 ng/ml in the blood samples collected during the winter or spring and 32 ng/ml in samples collected during the summer or fall. Nearly 13% (summer/fall) to 36% (winter/spring) of the control participants were deficient in 25(OH)D (<20 ng/ml) and 51% (summer/fall) and 77% (winter/spring) had insufficient plasma 25(OH)D levels (<32 ng/ml). Plasma levels of 1,25(OH)2D did not vary by season. Men whose levels for both 25(OH)D and 1,25(OH)2D were below (versus above) the median had a significantly increased risk of aggressive prostate cancer (odds ratio [OR] = 2.1, 95% confidence interval [CI] 1.2-3.4), although the interaction between the two vitamin D metabolites was not statistically significant (pinteraction = 0.23). We observed a significant interaction between circulating 25(OH)D levels and the VDR FokI genotype (pinteraction < 0.05). Compared with those with plasma 25(OH)D levels above the median and with the FokI FF or Ff genotype, men who had low 25(OH)D levels and the less functional FokI ff genotype had increased risks of total (OR = 1.9, 95% CI 1.1-3.3) and aggressive prostate cancer (OR = 2.5, 95% CI 1.1-5.8). Among men with plasma 25(OH)D levels above the median, the ff genotype was no longer associated with risk. Conversely, among men with the ff genotype, high plasma 25(OH)D level (above versus below the median) was related to significant 60% approximately 70% lower risks of total and aggressive prostate cancer.

CONCLUSIONS

Our data suggest that a large proportion of the US men had suboptimal vitamin D status (especially during the winter/spring season), and both 25(OH)D and 1,25(OH)2D may play an important role in preventing prostate cancer progression. Moreover, vitamin D status, measured by 25(OH)D in plasma, interacts with the VDR FokI polymorphism and modifies prostate cancer risk. Men with the less functional FokI ff genotype (14% in the European-descent population of this cohort) are more susceptible to this cancer in the presence of low 25(OH)D status.

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

The serine/threonine protein kinase Akt has recently been shown to be implicated in the pathway leading to cell survival in response to serum and growth factors in a variety of cellular systems. However, the existence of a biochemical route connecting this kinase to the large family of receptors that signal through heterotrimeric G proteins is yet to be explored. In this study, we set out to investigate whether GTP-binding protein (G protein)-coupled receptors (GPCRs) can stimulate Akt activity and survival pathways and, if so, to define the mechanism(s) whereby this class of cell surface receptors could regulate Akt function. Using ectopic expression of GPCRs in COS-7 cells as a model, we have observed that both m1 and m2 muscarinic acetylcholine receptors, representative of those GPCRs coupled to Gq and Gi proteins, respectively, can readily activate an epitope-tagged form of Akt kinase and prevent UV-induced apoptosis. We have also found that the pathway connecting G proteins to Akt implicates signals emanating from Galphaq, Galphai, and beta gamma dimers, but not from Galphas or Galpha12, in each case acting through a pathway that involves a phosphatidylinositol-3-OH kinase activity. Moreover, our findings suggest a role for a novel beta gamma-sensitive complex, p101.phosphatidylinositol-3-OH kinase-gamma, in the transduction of signals leading to Akt stimulation and cell survival by GPCRs and open new avenues for research on the function of the large family of G protein-linked receptors in the regulation of anti-apoptotic pathways.

BACKGROUND

Epidemiological studies have yielded inconsistent associations between vitamin D status and prostate cancer risk, and few studies have evaluated whether the associations vary by disease aggressiveness. We investigated the association between vitamin D status, as determined by serum 25-hydroxyvitamin D [25(OH)D] level, and risk of prostate cancer in a case-control study nested within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial.

METHODS

The study included 749 case patients with incident prostate cancer who were diagnosed 1-8 years after blood draw and 781 control subjects who were frequency matched by age at cohort entry, time since initial screening, and calendar year of cohort entry. All study participants were selected from the trial screening arm (which includes annual standardized prostate cancer screening). Conditional logistic regression was used to estimate adjusted odds ratios (ORs) with 95% confidence intervals (CIs) by quintile of season-standardized serum 25(OH)D concentration. Statistical tests were two-sided.

RESULTS

No statistically significant trend in overall prostate cancer risk was observed with increasing season-standardized serum 25(OH)D level. However, serum 25(OH)D concentrations greater than the lowest quintile (Q1) were associated with increased risk of aggressive (Gleason sum>> or = 7 or clinical stage III or IV) disease (in a model adjusting for matching factors, study center, and history of diabetes, ORs for Q2 vs Q1 = 1.20, 95% CI = 0.80 to 1.81, for Q3 vs Q1 =1.96, 95% CI = 1.34 to 2.87, for Q4 vs Q1 = 1.61, 95% CI = 1.09 to 2.38, and for Q5 vs Q1 = 1.37, 95% CI = 0.92 to 2.05; P(trend) = .05). The rates of aggressive prostate cancer for increasing quintiles of serum 25(OH)D were 406, 479, 780, 633, and 544 per 100 000 person-years. In exploratory analyses, these associations with aggressive disease were consistent across subgroups defined by age, family history of prostate cancer, diabetes, body mass index, vigorous physical activity, calcium intake, study center, season of blood collection, and assay batch.

CONCLUSIONS

The findings of this large prospective study do not support the hypothesis that vitamin D is associated with decreased risk of prostate cancer; indeed, higher circulating 25(OH)D concentrations may be associated with increased risk of aggressive disease.

The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues.

OBJECTIVE

Epidemiological evidence is suggestive, but limited, for an association between circulating 25-hydroxyvitamin D (25[OH]D) and risk of type 2 diabetes. We conducted a systematic review and meta-analysis that included new data from previously unpublished studies.

METHODS

Using a nested case-cohort design in the European Prospective Investigation into Cancer (EPIC)-Norfolk study, we identified a random subcohort and incident type 2 diabetes cases occurring between baseline (1993-1997) and 2006. In the Ely prospective study we identified incident type 2 diabetes cases between 1990 and 2003. We conducted a systematic review of prospective studies on 25(OH)D and type 2 diabetes published in MEDLINE or EMBASE until 31 January 2012, and performed a random-effects meta-analysis combining available evidence with results from the EPIC-Norfolk and Ely studies.

RESULTS

In EPIC-Norfolk, baseline 25(OH)D was lower among incident type 2 diabetes cases (mean [SD] 61.6 [22.4] nmol/l; n=621) vs non-case subcohort participants (mean 65.3 [23.9] nmol/l; n=826). There was an inverse association between baseline 25(OH)D and incident type 2 diabetes in multivariable-adjusted analyses: HR (95% CI) 0.66 (0.45, 0.97), 0.53 (0.34, 0.82), 0.50 (0.32, 0.76), p trend <0.001, comparing consecutive increasing 25(OH)D quartiles with the lowest. In Ely, 37 incident type 2 diabetes cases were identified among 777 participants. In meta-analysis, the combined RR of type 2 diabetes comparing the highest with lowest quartile of 25(OH)D was 0.59 (0.52, 0.67), with little heterogeneity (I (2) =2.7%, p=0.42) between the 11 studies included (3,612 cases and 55,713 non-cases).

CONCLUSIONS

These findings demonstrate an inverse association between circulating 25(OH)D and incident type 2 diabetes. However, causal inference should be addressed through adequately dosed randomised trials of vitamin D supplementation or genetic Mendelian randomisation experiments.

The evidence is compelling that free radicals, plus increases in free cytosolic Ca2+ and Na+, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca2+ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (.OH) radicals, plus ascorbyl and other carbon-centered radicals when exposed to 2.5 microM Ca2+, 14 mM Na+, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin-induced neurodegeneration. In a feed-forward cycle, exposure of isolated mitochondria to .OH significantly increases subsequent radical production five- to 16-fold (average = 8.8 +/- 1.6 SE, n = 6, p>> 0.01) with succinate as substrate, and also selectively impairs function of NADH-CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after .OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.

Interleukin-1 (IL-1) alpha and beta and tumor necrosis factor (TNF) alpha and beta are potent stimulators of bone resorption in vitro and in vivo. However, the mechanisms underlying this increased bone resorption have not been clearly defined. Increased bone resorption can result from increased activity of individual osteoclasts, increased numbers of osteoclasts, or both. Therefore, we have used a long-term human marrow culture system that forms multinucleated cells (MNC) with the characteristics of osteoclasts to examine the effects of IL-1 and TNF on osteoclast formation. Human recombinant IL-1 alpha and IL-1 beta, and human recombinant TNF-alpha and TNF-beta stimulated MNC formation from 4- to 60-fold. IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta significantly increased MNC formation at very low concentrations: 2.5 x 10(-13) M for IL-1 alpha and IL-1 beta, 10(-11) M for TNF-alpha, and 10(-10) M for TNF-beta In addition, these cytokines enhanced MNC formation in the presence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], a potent osteotropic factor that stimulates MNC formation by stimulating fusion of mononuclear precursor cells. Simultaneous addition of IL-1 and TNF to the cultures resulted in a synergistic stimulation of MNC formation. These results suggest that: (1) IL-1 and TNF stimulate bone resorption in part by increasing osteoclast formation and (2) an extremely low concentration of these factors can synergistically increase osteoclast formation in the absence of other factors, such as 1,25-(OH)2D3. These data suggest that synergistic interactions among cytokines play an important role in maintaining bone cell activity in normal and pathologic states.

BACKGROUND

The relationship between serum 25-hydroxyvitamin D [25(OH) vitamin D] concentration and hip fractures is unclear.

OBJECTIVE

To see whether low serum 25(OH) vitamin D concentrations are associated with hip fractures in community-dwelling women.

METHODS

Nested case-control study.

METHODS

40 clinical centers in the United States.

METHODS

400 case-patients with incident hip fracture and 400 control participants matched on the basis of age, race or ethnicity, and date of blood draw. Both groups were selected from 39 795 postmenopausal women who were not using estrogens or other bone-active therapies and who had not had a previous hip fracture.

METHODS

Serum 25(OH) vitamin D was measured and patients were followed for a median of 7.1 years (range, 0.7 to 9.3 years) to assess fractures.

RESULTS

Mean serum 25(OH) vitamin D concentrations were lower in case-patients than in control participants (55.95 nmol/L [SD, 20.28] vs. 59.60 nmol/L [SD, 18.05]; P = 0.007), and lower serum 25(OH) vitamin D concentrations increased hip fracture risk (adjusted odds ratio for each 25-nmol/L decrease, 1.33 [95% CI, 1.06 to 1.68]). Women with the lowest 25(OH) vitamin D concentrations (< or =47.5 nmol/L) had a higher fracture risk than did those with the highest concentrations >> or =70.7 nmol/L) (adjusted odds ratio, 1.71 [CI, 1.05 to 2.79]), and the risk increased statistically significantly across quartiles of serum 25(OH) vitamin D concentration (P for trend = 0.016). This association was independent of number of falls, physical function, frailty, renal function, and sex-steroid hormone levels and seemed to be partially mediated by bone resorption.

CONCLUSIONS

Few case-patients were nonwhite women. Bone mineral density and parathyroid hormone levels were not accounted for in the analysis.

CONCLUSIONS

Low serum 25(OH) vitamin D concentrations are associated with a higher risk for hip fracture.

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.

There is a high prevalence of chronic hyponatremia in the elderly, frequently owing to the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Recent reports have shown that even mild hyponatremia is associated with impaired gait stability and increased falls. An increased risk of falls among elderly hyponatremic patients represents a risk factor for fractures, which would be further amplified if hyponatremia also contributed metabolically to bone loss. To evaluate this possibility, we studied a rat model of SIADH and analyzed data from the Third National Health and Nutrition Examination Survey (NHANES III). In rats, dual-energy X-ray absorptiometry (DXA) analysis of excised femurs established that hyponatremia for 3 months significantly reduced bone mineral density by approximately 30% compared with normonatremic control rats. Moreover, micro-computed tomography (microCT) and histomorphometric analyses indicated that hyponatremia markedly reduced both trabecular and cortical bone via increased bone resorption and decreased bone formation. Analysis of data from adults in NHANES III by linear regression models showed that mild hyponatremia is associated with increased odds of osteoporosis (T-score -2.5 or less) at the hip [odds ratio (OR) = 2.85; 95% confidence interval (CI) 1.03-7.86; p < .01]; all models were adjusted for age, sex, race, body mass index (BMI), physical activity, history of diuretic use, history of smoking, and serum 25-hydroxyvitamin D [25(OH)D] levels. Our results represent the first demonstration that chronic hyponatremia causes a substantial reduction of bone mass. Cross-sectional human data showing that hyponatremia is associated with significantly increased odds of osteoporosis are consistent with the experimental data in rodents. Our combined results suggest that bone quality should be assessed in all patients with chronic hyponatremia.

Aminoacyl-RNA synthetases can be divided into two classes according to structural features inferred from sequence alignments. This classification correlates almost perfectly with the attachment of the amino acid to the 2'-OH (class I) or 3'-OH (class II) group of the terminal adenosine. Six subgroups of higher homology can be inferred from sequence analysis. The five aminoacyl-tRNA synthetases whose crystal structures are known (MetRS, TyrRS and GlnRS in class I, SerRS and AspRS in class II) belong to different subgroups. Two of them, GlnRS and AspRS, have been cocrystallized with their cognate tRNA. AspRS, like six other members of class II, is an alpha 2 dimer. Yeast tRNA(Asp) exhibits five identity determinants: the three anticodon bases, the discriminator base G73 and the base pair G10-U25. We report here that the refined crystal structure of AspRS complexed with tRNA(Asp) at 2.9 A resolution reveals three regions of contact, each involving a domain of AspRS and at least one identity determinant of tRNA(Asp). The mode of binding of the acceptor stem of tRNA(Asp) by AspRS can be generalized to class II aminoacyl-tRNA synthetases, whereas the deciphering of the anticodon, which involves a large conformational change of the loop and the formation of a bulge, is more specific to the aspartic system.

With the use of x-ray absorption spectroscopy, we have found that the Fe-O bond in chloroperoxidase compound II (CPO-II) is much longer than expected for an oxoiron(IV) (ferryl) unit; notably, the experimentally determined bond length of 1.82(1) A accords closely with density functional calculations on a protonated ferryl (Fe(IV)-OH, 1.81 A). The basicity of the CPO-II ferryl [pKa>> 8.2 (where Ka is the acid dissociation constant)] is attributable to strong electron donation by the axial thiolate. We suggest that the CPO-II protonated ferryl is a good model for the rebound intermediate in the P450 oxygenation cycle;with elevated pKa values after one-electron reduction, thiolate-ligated ferryl radicals are competent to oxygenate saturated hydrocarbons at potentials that can be tolerated by folded polypeptide hosts.

This study was designed to determine whether oxygen-derived free radicals play a role in the pathogenesis of gastric lesions produced by hypotensive ischemia in the rat. To achieve this goal, allopurinol, an inhibitor of xanthine oxidase (the enzyme responsible for the formation of superoxide radicals); superoxide dismutase, a scavenger of superoxide radicals (O2-); and dimethyl sulfoxide, a scavenger of hydroxyl radicals (OH) were used. In the anesthetized rat, HCl (0.1 N) was instilled into the pylorus-ligated stomach, and the rat was bled to reduce the blood pressure to less than 30 mmHg. The blood pressure was maintained at less than 30 mmHg for 20 min and then the shed blood was retransfused. Twenty minutes after the retransfusion the rat was killed, the stomach was removed, and the area of gastric mucosal lesions was measured. Both allopurinol and superoxide dismutase, but not dimethyl sulfoxide, significantly protected against hemorrhagic shock-induced gastric lesions. These findings suggest that oxygen-derived free radicals, particularly O2-, play an important role in the formation of gastric lesions produced by ischemia plus HCl.

BACKGROUND

The most reliable assessment of vitamin D status is measurement of plasma 25-hydroxyvitamin D (25[OH]D) concentration. High variability in 25(OH)D measurements due to utilized test and assay technologies and the lack of standardization against reference materials and reference method often confounds proper assessment of vitamin D status.

METHODS

We evaluated the accuracy of six routinely available methodologies: high-performance liquid chromatography (HPLC), the IDS-radioimmunoassay (IDS-RIA) and enzyme immunoassay (IDS-EIA), the Nichols Advantage automated protein-binding assay (Advantage), two versions of the DiaSorin automated immunoassay (Liaison 1 and Liaison 2)--and one prototype automated immunoassay (Elecsys) for assessment of the 25(OH)D(3) status in a cohort of 300 randomly selected patients' samples compared with the reference method liquid chromatography-tandem mass spectrometry (LC-MS/MS).

RESULTS

Passing-Bablok regression analysis demonstrated a slope for each method compared with LC-MS/MS that varied from 0.62 (IDS-EIA) to 1.0 (HPLC). The Advantage and the Liaison 1 showed significant deviation from linearity with highly variable individual results vs. the LC-MS/MS. Difference plots revealed a considerable persistent proportional bias for the IDS-RIA and IDS-EIA. All evaluated methods except HPLC demonstrated a more or less considerable deviation of individual 25(OH)D(3) values compared with LC-MS/MS defined target concentrations.

CONCLUSIONS

Standardization of methods for the quantification of 25(OH)D on a human-based sample panel by means of LCMS/MS would help to reduce the inter-method variability with respect to accuracy existing in 25(OH)D measurement considerably. However, there will still remain differences in the accuracy of methods utilizing sample purification before final quantification or immunological reaction when compared with those methods without separate sample purification.